This invention relates to nucleic acid and amino acid sequences of human hydrolase-like molecules and to the use of these sequences in the diagnosis, or treatment of cell proliferation disorders, and autoimmune disorders.
Hydrolysis is a common enzymatic mechanism. There are numerous enzymes whose catalytic mechanism involves breaking a covalent bond in a substrate by the addition of a molecule of water across the bond. The reaction involves a nucleophilic attack by the water molecule""s oxygen atom on a target bond within the substrate and results in a splitting of the water molecule across the target bond, thereby breaking the bond and generating two product molecules. This general mechanism applies to a wide variety of enzymes including phosphatases, lysophospholipases, glyoxalases, and phosphodiesterases.
The protein phosphorylation/dephosphorylation cycle is one of the major regulatory mechanisms employed by eukaryotic cells to control cellular activities. During protein phosphorylalion, phosphate groups are transferred from adenosine triphosphate molecules to a protein by protein kinases. During protein dephosphorylation, phosphate groups are removed from a protein by protein phosphatases using a hydrolytic mechanism. In this manner, phosphatases are involved in the control of many cellular signaling events that regulate cell growth and differentiation, cell-to-cell contact, the cell cycle, and oncogenesis. Protein phosphatases may be characterized by amino acid residue specificity, for example, serine/threonine phosphatases, tyrosine phosphatases, or dual-specificity phosphatases. Dual-specificity phosphatases contain a conserved cysteine residue essential for catalytic activity. (Ishibashi et al. (1992) Proc Natl Acad Sci 89:12170-12174.)
Lysophospholipases (LPLs) are widely distributed enzymes that regulate intracellular lipids, and occur in numerous isoforms. These isoforms vary in molecular mass, the substrate metabolized, and the optimum pH required for activity. Small isoforms, approximately 15-30 kDa, function as hydrolases; large isoforms, those exceeding 60 kDa, function both as hydrolases and transacylases. A particular substrate for LPLs, lysophosphatidylcholine, causes lysis of cell membranes when it is formed or imported into a cell. LPLs are regulated by lipid factors including acylcarnitine, arachidonic acid, and phosphatidic acid. Thus, the activity of LPLs is regulated by signaling molecules important in numerous pathways including the inflammatory response.
The glyoxylase system consists of glyoxylase I, which catalyzes the formation of S-D-lactoylglutathione from methyglyoxal, a side product of triose-phosphate energy metabolism, and glyoxylase II, which hydrolyzes S-D-lactoylglutathione to D-lactic acid and reduced glutathione. Methyglyoxal levels are elevated during hyperglycemia and are likely due to increased triose-phosphate energy metabolism. Elevated levels of glyoxylase II activity have been found in human and in a rat model of non-insulin-dependent diabetes mellitus. The glyoxylase system has been implicated in the detoxification of bacterial toxins and in the control of cell proliferation and microtubule assembly. Elevated levels of S-D-lactoylglutathione, the substrate of glyoxylase II, induced growth arrest and toxicity in HL60 cells. Thus, the glyoxylase system, and glyoxylase II in particular, may be associated with cell proliferation and autoimmune disorders such as diabetes.
Sphingomyelin is a membrane phospholipid that is hydrolyzed to ceramide and phosphatidylcholine by the action of the phosphodiesterase, acid sphingomyelinase. Phosphatidylcholine is involved in numerous intracellular signaling pathways, while ceramide is an essential precursor for the generation of gangliosides, membrane lipids found in high concentration in neural tissue. Defective acid sphingomyelinase phosphodiesterase leads to a build-up of sphingomyelin molecules in lysosomes, resulting in Niemann-Pick disease.
Post-translation chemical modification of proteins, such as phosphorylation by protein kinase A, has been demonstrated to affect biochemical activity of enzymes (Krebs (1981) Curr Top Cell Regul 18:401-419). In a further example, protein kinase C a phosphorylates and activates the growth promoter Raf (Kolch et al. (1993) Nature 364: 249-252).
The discovery of new human hydrolase-like molecules and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, or treatment of cell proliferation disorders and autoimmune disorders.
The invention features substantially purified polypeptides, human hydrolase-like molecules, referred to collectively as xe2x80x9cHHLMxe2x80x9d and individually as xe2x80x9cHHLM 1-8xe2x80x9d. In one aspect, the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-8 or portions or fragments thereof.
The invention further provides a substantially purified variant having at least 81% amino acid identity to the amino acid sequences of SEQ ID NOs:11-8 or fragments thereof. The invention also provides an antigenic epitope selected from SEQ ID NOs:1-8, an oligopeptide selected from of SEQ ID NOs:1-8, and a biologically active portion selected from SEQ ID NOs:1-8. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-8 or fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-8 or fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOs:1-8 or fragments thereof. The invention also provides a purified polypeptide of an amino acid sequence encoded by a polynucleotide selected from SEQ ID NOs:17-55.
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs:9-16 or fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs:9-16 or fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs:9-16 or fragments thereof
The invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs:17-55 or fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs:17-55 or fragments thereof, as well as an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NOs:17-55 or fragments thereof
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:1-8 or fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NOs:1-8 or fragments thereof, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified HHLM having the amino acid sequence of SEQ ID NOs:1-8 or fragments thereof in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the amino acid sequence of SEQ ID NOs:1-8 or fragments thereof, as well as a purified agonist and a purified antagonist to the polypeptide.
The invention also provides a method for treating or preventing a cell proliferation disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence of SEQ ID NOs:1-8 or fragments thereof.
The invention also provides a method for treating or preventing an autoimmune disorder, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having an amino acid sequence of SEQ ID NOs:1-8 or fragments thereof.
The invention also provides a method for detecting a polynucleotide encoding the polypeptide comprising the amino acid sequence SEQ ID NOs:1-8 or fragments thereof in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence encoding the polypeptide comprising SEQ ID NOs:1-8 or fragments thereof to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.
The invention further provides a method of using a polypeptide to screen a plurality of molecules or compounds to identify a ligand which specifically binds the polypeptide. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs.
The invention further provides a method of using a polypeptide to screen subject sample for antibodies which specifically bind the polypeptide.
The invention further provides a method of using a polypeptide to prepare and purify antibodies which specifically bind the polypeptide.